The present invention relates to gene expression levels and, more particularly, to the determination of gene expression levels using nucleic acid microarrays.
The study of gene expression levels is a focus of current research. Gene expression analysis may be carried out using microarrays. Microarrays allow for the rapid analysis of large quantities of genetic material. For a detailed discussion of microarrays, see P. O. Brown, et al., xe2x80x9cExploring the New World of the Genome With DNA Microarrays,xe2x80x9d Nature Genetics v. 21, p. 33 (1999); R. J. Lipshutz, et al., xe2x80x9cHigh Density Synthetic Oligonucleotide Arrays,xe2x80x9d Nature Genetics v. 21, p. 20 (1999), the disclosures of which are incorporated by reference herein. Microarrays allow researchers to analyze the genetic information in cells, e.g., genes, to determine whether each gene is being expressed in the cell, and if so, to quantify the expression level. Since gene expression levels play a central role in determining the phenotype, the observable characteristics of the cell, the gene expression levels play a crucial part in diagnosing disease states of the cell (for example, identifying cells that are, or likely to become, cancerous), in understanding cell cycles and differentiation and in casting light upon the complex spectrum of behaviors and changes in a cell.
Microarray technologies take several different forms. Some microarrays contain billions of relatively short oligonucleotides, i.e., less than 100 base pairs, of single DeoxyriboNucleic Acid (DNA) strands, termed xe2x80x9cprobe oligonucleotides,xe2x80x9d bonded to a substrate. Each of the probe oligonucleotides represents an xe2x80x9cunzippedxe2x80x9d piece of a gene available for binding with a complementary strand to which the probe oligonucleotide can be xe2x80x9czipped.xe2x80x9d Ideally, many identical copies of each probe oligonucleotide are deposited onto an area, i.e., about 20 microns square, termed a xe2x80x9cprobe spot,xe2x80x9d on the substrate. A collection of probe spots on a microarray can represent thousands of genes.
Oligonucleotide sequences which are representative of the genes of interest within a cell are called target oligonucleotides. The concentration of target oligonucleotides is a measure of gene expression levels. A number of target oligonucleotides may be complementary to the probe oligonucleotides. Each target oligonucleotide has a strong affinity for bonding, i.e., hybridizing, with the probe oligonucleotide to which it is complementary. Thus, once a solution containing target oligonucleotides is introduced into the microarray, those probe oligonucleotides of the microarray that are complementary, or nearly complementary, to target oligonucleotides in the solution will hybridize with those target oligonucleotides, producing probe spots of the microarray containing hybridized probe oligonucleotide and target oligonucleotide double strands. Other spots wherein the probe oligonucleotides are not complementary to any target oligonucleotides in the solution will contain only the unhybridized, single-stranded probe oligonucleotides. Fluorescent tags on the target oligonucleotides allow for the detection of probe spots containing hybridized probe oligonucleotides and target oligonucleotides. Thus, the target oligonucleotides present in the solution may be determined. Moreover, the intensity of the fluorescence provides a measure of how much of each target oligonucleotide is present in the target solution, and hence in the original cell.
Gene expression analysis using conventional methods, such as the Affymetrix(copyright) Genechips(copyright) and algorithms has several shortcomings. For example, in the Affymetrix(copyright) technology, each gene is typically represented by a plurality of different probes of short length, i.e., about 25 nucleotide base pairs. Since all the target oligonucleotides representing a given gene are present in roughly equal concentrations, measured intensity values of all the probe spots corresponding to that gene are expected to be virtually identical. In fact, however, there are typically sizable variations in measured hybridization intensity values among the probe spots representing a particular gene. These variations may be due to, for example, cross-hybridization, secondary structure (hybridization of segments of a single target oligonucleotide or probe oligonucleotide with one other), or stronger tendency for certain complementary pairs to hybridize than for others. Variations greatly complicate the task of determining a unique and correct expression level for each gene in the target solution.
Thus, there exists a need for an accurate and efficient technique for determining gene expression levels.
The present invention provides techniques for analyzing gene expression. In one aspect of the invention, the technique provides a method for determining a concentration level of a target nucleic acid, the target nucleic acid comprising at least one target oligonucleotide. The method determines (i) a measure of affinity value of the target oligonucleotide with a probe oligonucleotide; and (ii) a hybridization intensity value for the target oligonucleotide and the probe oligonucleotide at a probe spot. The measure of affinity value and the hybridization intensity value are used to determine the concentration level of the target nucleic acid.
The measure of affinity value may comprise a free energy of hybridization of the target oligonucleotide with the probe oligonucleotide. Further, the measure of affinity value may comprise a net rate of hybridization of the target oligonucleotide with the probe oligonucleotide.
A more complete understanding of the present invention, as well as further features and advantages of the present invention, will be obtained by reference to the following detailed description and drawings.